miRNA and proteins in Neuroblastoma tumorigenesis: perspectives analyses on miR34a function

Neuroblastoma is a childhood cancer that originates from precursor cell of sympathetic nervous system, occurring primarily in children under the age of 5 years. The disease is highly heterogeneous, ranging from spontaneous regression to rapid progression with high death rate in pediatric oncology. Expression profiling studies of Neuroblastoma primary tumors have identified many miRNAs whose expression levels have been significantly associated with poor patient survival. MiR-34a is a member of an evolutionarily conserved miRNA family, miR-34s. Mir34a functions as tumor suppressor in Neuroblastoma and the inactivation and absence of miR-34a has been shown related to the pathogenesis of a variety of tumors. Several genes have been shown to be direct miR-34a targets, encoding for proteins involved in proliferation, invasion and apoptosis. However, it is likely that miR-34a regulates many additional, as yet unconfirmed targets, since bioinformatic predictions suggest that several hundred of mRNAs contain matches to the miR-34a seed sequence. To address this question, here, we describe a global analysis of the effect of miR-34a expression at early time points (6 and 12 hrs) on proteome using a shotgun analysis with post-metabolic differential labeling in two different aggressive Neuroblastoma cell lines. This technology allow us to identify 2082 proteins, of which 172 were found differentially regulated, with 113 proteins being down- and 68 up-regulated. Computational analyses were employed to assign to each identified peptide its protein name and its specific level of regulation. When considering protein with a predicted seed-matching site among the protein identified, we found that only 33 proteins retain seed sequence in the 3’ UTR of the corresponding mRNA, as predicted by PITA tool. By combining the results generated by shotgun and Kaplan Meier for overall survival analyses, we found seven proteins/genes correlated to worse clinical outcome when overexpressed in NBL. These genes are involved in the differentiation (LYAR, CTCF), apoptosis and proliferation (TGM2, Ki67), molecular transport (TIMM13 and ABCF2) and several metabolic pathway (ALG13). Furthermore, these proteins are strongly linked each other and their impairment affects several pathways, including those signaling pathways such as TGF-β, Wnt and Src. Here, we comprehensively demonstrate the capabilities of miR-34a to target simultaneously components of several cancer signaling cascade, involved in Neuroblastoma progression. The level of this analysis was able to deeply understand which other protein are targeted and at what specific time of action are then down-regulated by miR34a, given a second level of target identification and showing the importance of this action in Neuroblastoma as one of the prominent phenomena occur to counteract the progression of tumorigenesis. Moreover, we demonstrated that the magnitude of miR-34a effect on protein expression changes occurs at early time when compared to the later proteome output. Finally, to assess trend analysis, of the proteins differentially expressed at different time points (6h, 12h and 24h), Lorenz curve and Gini coefficient were evaluated for each protein, observing that several proteins were dinamically regulated and highlighting that machinery involving protein downregulation, by miRNA, are not linear. Our data along with previous studies strongly suggest the potential therapeutic usefulness of miR-34a that could offer better chance of therapeutic success.